The goal of this project is the development of cell separation methods for the specific isolation of immune cells, particularly for varieties of antigen-reactive cells involved in cellular immune reactions, and for their subcellular fractionation in order to study the mechanisms involved in the development of immune reactivities and immune macromolecules. Antigen-reactive lymphocytes (ARC) are obtained from the spleens of normal mice and from mice immunized with allogeneic tumor cells and normal cells. Spleen cell suspensions containing ARC are tested for binding to the cell surface antigens of target cells attached to insoluble supports. The nonadherent cells are tested for depletion of cytotoxic effector activity by measurement of 51Cr release from target cells. Graft-versus-host (GVH) activity is measured by the Simonsen spleen weight assay in neonatal mice. The mixed lymphocyte culture is used to test for stimulation of ARC by alloantigen in vitro, and for the generation of cytoxic effector cells from their precursors.